Cultured human lymphoblasts (CCRF-CEM) depleted of intracellular thymidine nucleotides by the antifolate, methotrexate, incorporate (3H)thymidine at full specific activity allowing the detection of early intermediates of DNA replication. A short pulse of (3H) thymidine is incorporated first into short polynucleotide chains, with a mean chain length of about 75 nucleotides, which during further incubation are converted to longer DNA molecules. Nucleic acids isolated from the cells pulse labeled with either (3H)thymidine or (3H)uridine contain RNA that is associated with some of the nascent DNA in Cs2SO4 equilibrium density gradients. Moreover, a small amount of the (3H) labeled RNA remained bound to DNA after repeated denaturation and partial purification by equilibrium centrifugation and column chromatography. Treatment of the (3H)labeled RNA with alkali or RNase rendered the radioactivity acid-soluble showing that this material is mainly RNA. These results are consistent with the possibility that the RNA is covalently linked to some of the nascent DNA, and may act as a primer for DNA chain initiation in these cells. BIBLIOGRAPHIC REFERENCES: Fridland, A.: Differential effect of 1-beta-D-arabinofuranosylcytosine (ARA-C) on DNA chain initiation and elongation in human lymphoblasts. Proc. Amer. Assoc. Cancer Res. 18:00, 1977. Fridland, A.: Synthesis of small polynucleotide chains in human lymphoblasts pretreated with methotrexate: A possible mechanism for DNA chain growth. Adv. Enz. Regu. 15:00, 1977. (Editor, G. Weber), Pergamon Press, New York.